Adipose-Derived Stem Cells as a Tool for Dental Implant Osseointegration: an Experimental Study in the Dog.

The biological interaction between the jaw bones and dental implant is fundamental for the long-term success of dental implant placement. Nevertheless, the insufficient bone volume remains a major clinical problem, especially in case of immediate dental implant. Using a canine model, the present study proves the regenerative potential of adipose- derived stem cells (ADSCs) to repair peri-implant bone defects occurring in immediate dental implant placement. In six labradors, all mandibular premolars and the first molars were extracted bilaterally and three months later dental implants were installed with a marginal gap. The marginal defects were filled with hydroxyapatite (HA)-based scaffolds previously seeded with ADSCs. After one month of healing, specimens were prepared for histological and histomorphometric evaluations. Histological analyses of ground sections show that ADSCs significantly increase bone regeneration. Several new vessels, osteoblasts and new bone matrix were detected. By contrast, no inflammatory cells have been revealed. ADSCs could be used to accelerate bone healing in peri- implant defects in case of immediate dental implant placement.

he osseointegration is the direct rigid fixation of the dental implant into jaw bones (1). The long-term success of implant-supported protheses is ensured by the biological interaction between the jaw bones and dental implant surfaces. As implant surface properties strongly influence the bone response, so various techniques of surface treatment have been developped and applied to improve implant stability during bone regeneration (2)(3)(4).
Nevertheless, a great clinical problem especially in case of immediate dental implant placement is represented by the insufficient bone volume (5).
Immediate implant offers several advantages compared to the delayed implant, such as time reduction for making dentures, which may lead to an immediate satisfaction regarding aesthetics and T Submmited 30 July 2015; Accepted 2 September 2015; Published 20 September 2015 function in patients (6). However, a significant alveolar bone loss is often present as an immediate post-extraction event (5). Several strategies could be applied in this case. The gold standard is represented by autologous bone grafts, but their clinical applications are very limited (7). In this view, the use of stem cells could provide a promising approach for enhancing osseointegration of immediate dental implants, especially for defects in which spontaneous repair is not workable (8)(9)(10)(11)(12). Therefore, a different cell source that possesses the same beneficial functions, while simultaneously overcoming their disadvantages, is adipose tissue (17)(18). Human adipose tissue can be obtained in larger quantities compared to the more invasive procedure for isolating BMSCs offering moreover little patient discomfort (19)(20). Stem cells isolated from adipose tissue are called (ADSCs) and have the same biological properties and characteristics of BMSCs. Indeed, ADSCs express the same cell surface markers and have the same differentiation potentials of BMSCs. Moreover, starting from the same raw material, the quantity of stem cells isolated are bigger from adipose tissue than from other sources, indicating that ADSC could be an important tool for use in regenerative medicine (20)(21)(22) (23). In this study, we aimed to apply our knowledge in bone tissue engineering in the restoration of peri-implant bone defects occurring in immediate dental implant placement procedures.
For this purpose, we have prepared bone substitutes cultivating ADSCs on HA-based scaffolds up to 28 days. Then, we have tested these constructs in vivo on a canine model of immediate dental implant placement.

Biomaterial and ADSCs seeding
The HA-based scaffolds were supplied in granules (Bio-Oss®, spongious bone substitute, Geistlich Pharma AG, Wolhusen, Switzerland). The granules were coated with 50 mg/mL fibronectin (Sigma-Aldrich) at room temperature for 4 h, then air-dried overnight in a sterile biosafety cabinet.
The HA granules were placed in a 1x10 6 ADSCs suspension under vacuum conditions for 60 seconds in order to facilitate the cells flow inside the pores.
After 2 h of incubation at 37 °C with 5% CO 2 , the scaffolds were cultured in cDMEM up to 28 days, and the medium was changed twice a week.

MTT assay
To determine the proliferation rate of cells grown on HA-based scaffolds, the MTT-based (methyl thiazolyl-tetrazolium) cytotoxicity assay was performed according to the method of Denizot and Lang with minor modifications (24). The test is based on mitochondria viability, i.e., only functional mitochondria can oxidize an MTT solution, giving a typical blue-violet end product.
After harvesting the culture medium, the cells were

Nuclear staining
The ADSCs distribution inside the HA-based scaffolds was detected by nuclear staining with Hoechst (H33342, Sigma-Aldrich). The samples were fixed in 4% phosphate-buffered formalin, pH 7, at room temperature for 10 min. After a wash in PBS, cells were stained with 2 µg/mL Hoechst solution in PBS for 5 min.

Scanning electron microscopy (SEM)
The HA-based scaffolds seeded with ADSCs were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at room temperature for 60 min.   (Fig. 2A). This behavior is also observed in SEM images where ADSCs appeared with a spindle shape in a continuous cell layer (Fig. 2B).   MSCs phenotype were investigated. The real-time PCR showed a well-defined expression for CD73, CD90 and CD105, whereas no expression for CD34 has been revealed (Fig. 3).  Fig. 4B). In both conditions, new ECM rich in collagen fibers has been deposited (black asterisks in Fig. 4C and Fig. 4D). In particular, the HA-based scaffold enriched with ADSCs showed the presence of several vessels (black arrows in Fig 4C and Fig. 4E) embedded in the new ECM; on the contrary, vessels could not be detected in the HAbased scaffold alone ( Fig.4D and Fig. 4F). In the test condition, a regenerated bone tissue (dark red in Fig.   4C) was present at interface between implant and HA-based scaffold (white arrows in Fig. 4C).

Dental implant placement in the canine model
Presumably, the presence of ADSCs has considerably stimulated the osteogenic population (black arrowheads in Fig. 4E) mainly around the HA-based scaffolds (light red in Fig. 4E) to produce new bone matrix (dark red in Fig. 4E). Conversely, in the control condition, an ECM of collagen fibers (black asterisks in Fig 4D) was lay down at interface between implant and HA-based scaffold (white arrows in Fig. 4D), however, osteogenic cells were not detected (Fig. 4F). The destiny of ADSCs has been followed by immunofluorescence analysis.  Table 2. Evidently, the bigger values on NBF are in the site on which ADSCs are present.

Discussion
Often, the installation of dental implants is hampered by alveolar bone loss, especially in immediate implant placement. In order to reduce  Afterw-ards, several research has demontrated that MSC treatment could be very useful for bone repair (31). Table 1. Histomorphometric analysis of defects filled with HA-based scaffolds alone or previously seeded with ADSCs for 28 days.

HA-based scaffolds with ADSCs
Polymorphic nuclear cells a --

New bone + ++
Cells were scored from not present (-) to abundantly present (+++). a Polymorphic nuclear cells include i.e. granulocytes; b Phagocytic cells include macrophages and monocyte-derived giant cells; c Non-phagocytic cells include lymphocytes, plasma cells and mast cells.  Thus, bone regeneration process observed in our experimental data, is mediated by the transplanted ADSCs that may act by starting to recruit endogenous stem cell by means of trophic factor secretion without triggering any inflammatory response (38). In conclusion, our HA-based scaffold enriched with ADSCs could be a feasible and effective engineered bone tissue to use to accelerate bone healing in periimplant defects.